Novel therapeutic strategies for injured endometrium: intrauterine transplantation of menstrual blood­derived cells from infertile patients.

BACKGROUND: Menstrual blood-derived cells show regenerative potential as a mesenchymal stem cell and may therefore be a novel stem cell source of treatment for refractory infertility with injured endometrium. However, there have been few pre-clinical studies using cells from infertile patients, which need to be addressed before establishing an autologous transplantation. Herein, we aimed to investigate the therapeutic capacity of menstrual blood-derived cells from infertile patients on endometrial infertility. METHODS: We collected menstrual blood-derived cells from volunteers and infertile patients and confirmed their mesenchymal stem cell phenotype by flow cytometry and induction of tri-lineage differentiation. We compared the proliferative and paracrine capacities of these cells. Furthermore, we also investigated the regenerative potential and safety concerns of the intrauterine transplantation of infertile patient-derived cells using a mouse model with mechanically injured endometrium. RESULTS: Menstrual blood-derived cells from both infertile patients and volunteers showed phenotypic characteristics of mesenchymal stem cells. In vitro proliferative and paracrine capacities for wound healing and angiogenesis were equal for both samples. Furthermore, the transplantation of infertile patient-derived cells into uterine horns of the mouse model ameliorated endometrial thickness, prevented fibrosis, and improved fertility outcomes without any apparent complications. CONCLUSIONS: In our pre-clinical study, intrauterine transplantation of menstrual blood-derived cells may be a novel and attractive stem cell source for the curative and prophylactic therapy for injured endometrium. Further studies will be warranted for future clinical application.

Multicenter, 2-dose single-group controlled trial of tacrolimus for the severe infertility patients.

INTRODUCTION: Infertility is estimated to affect 8% to 12% of reproductive-aged couples worldwide. While approximately 85% of infertile couples have an identified cause, the remaining 15% suffer physically and emotionally from unexplained intractable infertility. In recent years, maternal-to-fetal immunological abnormalities have attracted attention as mechanisms that differ from the conventional factors contributing to infertility and pregnancy loss. A T-helper 2 (Th2)-dominant immune state has been proposed as a maternal immune alteration to eliminate rejection and induce tolerance to a semi-allogeneic fetus. An imbalance in Th1 responses would not induce adequate maternal immune tolerance to the fetus or early embryos. Tacrolimus, widely used as an immunosuppressant agent in solid organ transplant recipients, is expected to suppress maternal rejection and promote tolerance to early embryos after assisted reproductive technology by modulating the immunological environment of the preimplantation endometrium. We planned an exploratory clinical trial to determine the efficacy, safety, and dosage of tacrolimus in women with intractable infertility. METHODS AND ANALYSIS: This is a multicenter, 2-dose, single-group controlled trial in infertile women who failed to achieve a chemical pregnancy despite multiple in vitro fertilization (IVF) and embryo transfer (ET) treatment cycles. The following 2 key selection criteria were set: no underlying factors of infertility despite appropriate evaluation and presence of Th1-dominant immune state, defined as a Th1/Th2 cell ratio ≥ 10.3 in the peripheral blood. A total of 26 eligible participants are randomly assigned (in a 2:1 ratio) to receive immunosuppressive therapy with oral tacrolimus at a daily dose of 2 mg or 4 mg. Tacrolimus is administered for 16 days starting from 2 days before ET. The primary endpoint is the presence of clinical pregnancy 3 weeks after IVF/ET treatment, and the secondary endpoint is the presence of biochemical pregnancy 2 weeks after IVF/ET treatment. Safety evaluation and biomarker discovery for tacrolimus treatment in infertile women will be conducted simultaneously. TRIAL REGISTRATION NUMBER: Japan Registry of Clinical Trials (jRCT; jRCTs031220235).

Trehalose Suppresses Lysosomal Anomalies in Supporting Cells of Oocytes and Maintains Female Fertility.

Supporting cells of oocytes, i.e., cumulus cells, control oocyte quality, which determines fertilization success. Therefore, the transformation of mature and immature cumulus cells (MCCs and ICCs, respectively) into dysmature cumulus cells (DCCs) with dead characteristics deteriorates oocyte quality. However, the molecular basis for this transformation remains unclear. Here, we explored the link between autophagic decline and cumulus transformation using cumulus cells from patients with infertility, female mice, and human granulosa cell-derived KGN cell lines. When human cumulus cells were labeled with LysoTracker probes, fluorescence corresponding to lysosomes was enhanced in DCCs compared to that in MCCs and ICCs. Similarly, treatment with the autophagy inhibitor chloroquine elevated LysoTracker fluorescence in both mouse cumulus cells and KGN cells, subsequently suppressing ovulation in female mice. Electron microscopy analysis revealed the proliferation of abnormal lysosomes in chloroquine-treated KGN cells. Conversely, the addition of an autophagy inducer, trehalose, suppressed chloroquine-driven problematic lysosomal anomalies and ameliorated ovulation problems. Our results suggest that autophagy maintains the healthy state of the supporting cells of human oocytes by suppressing the formation of lysosomes. Thus, our results provide insights into the therapeutic effects of trehalose on female fertility.

Successful Pregnancy in a Case of Behçet's Disease after Treatment with Prednisolone.

A 34-year-old woman (gravida 1, para 0) visited the Division of Reproductive Medicine/National Center for Child Health and Development due to infertility; she had also been suffering from incompletely treated genital ulcers and stomatitis for 10 years. This case was diagnosed as an incomplete-type Behçet's disease (BD) at the Department of Maternal-Fetal Biology/National Center for Child Health and Development. Since no apparent abnormality was found in the general infertility test, artificial insemination with the husband's semen (AIH) was performed for the patient with unexplained infertility, which failed. However, after treating BD with prednisolone, chronic inflammation (stomatitis and genital ulcer) and immunological abnormalities (Th2 and NK cell activity) improved, and conception was possible by AIH. Thus, prednisolone administration may have induced immune tolerance in the patient with BD, which may have contributed to the success of AIH.

Membrane protein CD9 is repositioned and released to enhance uterine function.

Tetraspanin CD9 is essential for sperm-egg fusion and also contributes to uterine repair through microexosome formation. Microexosomes share CD9 with exosomes and are released from eggs and uterine epithelial cells. However, the mechanism for the formation of microexosomes remains unknown. To address this issue, we examined membrane localization and extracellular release of CD9 proteins using uterine epithelial cells and secretions in mice and humans. In mice, CD9 localized predominantly on the basal region of the plasma membrane and relocated to the apical region upon embryo implantation. Furthermore, extracellular CD9 proteins were detected in uterine secretions of mice and women undergoing infertility treatment, but were below detectable levels in supernatants of pluripotent stem cells. Ultrastructural analysis demonstrated that membrane projections were shortened and the number of mitochondria was reduced in uterine epithelial cells lacking Cd9 genes. Our results suggest that CD9 repositioning and release affect both membrane structures and mitochondrial state in the uterus, and contribute to female fertility.

Advanced paternal age alone does not adversely affect pregnancy or live-birth rates or sperm parameters following intrauterine insemination.

PURPOSE: This study aimed to evaluate the effect of advanced paternal age on pregnancy outcomes and sperm parameters following intrauterine insemination (IUI). We used IUI data rather than assisted reproductive technology data, which might mask the effects of sperm impairments. METHODS: We retrospectively analyzed 1576 IUI cycles in women under 40 years old between April 2012 and May 2016 at the National Center for Child Health and Development in Japan. The main outcomes were clinical pregnancy and live birth. RESULTS: The mean male age was significantly lower in cycles that resulted in pregnancy compared with those without pregnancy (38.0 vs 39.1 years; P < 0.001), with a similar trend for live-birth cycles. However, there was no relationship between advanced paternal age and pregnancy outcomes after adjusting for confounding factors and correlations within patients using generalized estimating equations, and the age of the female partner was the only factor affecting pregnancy rate. Furthermore, advanced paternal age had no effect on sperm parameters. CONCLUSIONS: Advanced paternal age alone does not adversely affect pregnancy or live-birth rates or sperm parameters following IUI.

Autophagy-disrupted LC3 abundance leads to death of supporting cells of human oocytes.

Autophagic recycling of cell parts is generally termed as the opposite of cell death. Here, we explored the relation between cell death and autophagy by examining granulosa cell layers that control oocyte quality, which is important for the success of fertilization. Granulosa cell layers were collected from infertile women and morphologically divided into four types, viz., mature (MCCs), immature (ICCs), and dysmature cumulus cells (DCCs), and mural granulosa cells (MGCs). Microtubule-associated protein light chain 3 (LC3), which is involved in autophagosome formation, was expressed excessively in DCCs and MGCs, and their chromosomal DNA was highly fragmented. However, autophagy initiation was limited to MGCs, as indicated by the expression of membrane-bound LC3-II and autophagy-related protein 7 (ATG7), an enzyme that converts LC3-I to LC3-II. Although pro-LC3 was accumulated, autophagy was disabled in DCCs, resulting in cell death. Our results suggest the possibility that autophagy-independent accumulation of pro-LC3 proteins leads to the death of human granulosa cells surrounding the oocytes and presumably reduces oocyte quality and female fertility.

Degradation of phosphate polymer polyP enhances lactic fermentation in mice.

In bacteria, a polymer of inorganic phosphate (Pi) (inorganic polyphosphate; polyP) is enzymatically produced and consumed as an alternative phosphate donor for adenosine triphosphate (ATP) production to protect against nutrient starvation. In vertebrates, polyP has been dismissed as a "molecular fossil" due to the lack of any known physiological function. Here, we have explored its possible role by producing transgenic (TG) mice widely expressing Saccharomyces cerevisiae exopolyphosphatase 1 (ScPPX1), which catalyzes hydrolytic polyP degradation. TG mice were produced and displayed reduced mitochondrial respiration in muscles. In female TG mice, the blood concentration of lactic acid was enhanced, whereas ATP storage in liver and brain tissues was reduced significantly. Thus, we suggested that the elongation of polyP reduces the intracellular Pi concentration, suppresses anaerobic lactic acid production, and sustains mitochondrial respiration. Our results provide an insight into the physiological role of polyP in mammals, particularly in females.

Birthweights and Down syndrome in neonates that were delivered after frozen-thawed embryo transfer: The 2007-2012 Japan Society of Obstetrics and Gynecology National Registry data in Japan.

Aim: To evaluate the use of frozen embryos on the outcome of assisted reproductive technology (ART), a retrospective study of the Japanese Assisted Reproductive Technology Registry data during the years 2007-2012 was conducted. Methods: A total of 124 946 singleton neonates who reached term gestation following ART from 2007-2012, with 80 660 achieved through frozen-thawed embryo transfer (ET) and 44 286 being achieved through fresh ET, were analyzed for their birthweights and chromosomal abnormalities. Results: The birthweight of the neonates from the frozen-thawed ETs was significantly higher than that of those from the fresh ETs throughout all the study years. The frequency of Down syndrome was 0.17% for the fresh ETs and 0.13% for the frozen-thawed ETs in the period 2007-2012. This study showed that frozen-thawed ETs result in a constant increase of the average birthweight between 37 and 41 weeks gestational age and lower frequencies of Down syndrome. Conclusion: Frozen-thawed ETs were comparable to the fresh ET method, with the exceptions of higher birthweights and a lower frequency of Down syndrome in the neonates that were born from frozen-thawed ET. The increase in birthweights was not proportional to the gestational ages. This cannot be explained with any well-known mechanism. The frequency of chromosomal abnormalities needs detailed data for analysis.

Increased incidence of post-term delivery and Cesarean section after frozen-thawed embryo transfer during a hormone replacement cycle.

PURPOSE: This study aimed to clarify the risks of adverse pregnancy outcomes in patients who conceive singletons after frozen embryo transfer (FET) during a hormone replacement cycle and their offspring. METHODS: A retrospective cohort study was conducted in patients who conceived after FET, based on the Japanese-assisted reproductive technology registry for 2013. The perinatal outcomes in cases with live-born singletons achieved through natural ovulatory cycle FET (NC-FET) (n = 6287) or hormone replacement cycle FET (HRC-FET) (n = 10,235) were compared. Multiple logistic regression analyses were performed to determine the potential confounding factors. RESULTS: The frequencies of macrosomia (1.1% in NC-FET and 1.4% in HRC-FET; P = 0.058) were comparable between patients after NC-FET and HRC-FET. The proportions of post-term delivery (0.2% in NC-FET and 1.3% in HRC-FET; P < 0.001) and Cesarean section (33.6% in NC-FET and 43.0% in HRC-FET; P < 0.001) were higher in patients after HRC-FET than in patients after NC-FET. The risks of post-term delivery (adjusted odds ratio (AOR) 5.68, 95% confidence interval (CI) 3.30-9.80) and Cesarean section (AOR 1.64, 95% CI 1.52-1.76) were also higher in patients after HRC-FET than in patients after NC-FET. CONCLUSIONS: Patients who conceived singletons after HRC-FET were at increased risk of post-term delivery and Cesarean section compared with those who conceived after NC-FET.

Changes in serum iodine concentration, urinary iodine excretion and thyroid function after hysterosalpingography using an oil-soluble iodinated contrast medium (lipiodol).

OBJECTIVE: Reports of hypothyroidism after hysterosalpingography (HSG) using lipiodol are emerging. The present study was designed to investigate the changes in serum iodine concentration (SIC), urinary iodine concentration/creatinine excretion (UI/Cr), and thyroid function before and after HSG using lipiodol. METHODS: The prospective observation study included 22 infertile euthyroid women with no previous history of thyroid disease. All underwent HSG between April 2007 and August 2008 at our institution. We examined SIC, UI/Cr, and thyroid function before HSG, and at 4, 8, 12, and 24 weeks, and 9-12 months after HSG. RESULTS: The median value of SIC and UI/Cr peaked at 4 weeks after HSG and remained at significantly high levels at 8, 12, and 24 weeks post-HSG compared with pre-HSG. In sync with the increase of iodine, the mean level of TSH significantly increased at 4, 8, 12, and 24 weeks post-HSG compared with pre-HSG. After 24 weeks, differences in SIC, UI/Cr, and TSH levels before and after HSG became nonsignificant. The mean value of free triiodothyronine and free thyroxine showed no significant difference at any of the time points compared with pre-HSG. Three cases (13.6%) showed transient high TSH (>5 µIU/L) with normal thyroid hormones at 4 or 8 weeks after HSG. CONCLUSION: Thyroid monitoring should be conducted in the first 4-8 weeks after HSG using lipiodol and attention to thyroid dysfunction should be paid for up to 6 months after the procedure due to the possibility of excess iodine.

Optimization of a novel nylon mesh container for human embryo ultrarapid vitrification.

OBJECTIVE: To evaluate the efficacy of a nylon mesh container in vitrification of human embryos and to determine the optimal osmotic pressure of the initial thawing solution. DESIGN: Retrospective analysis. SETTING: National Center for Child Health and Development, Tokyo, Japan. PATIENT(S): Infertile patients undergoing either in vitro fertilization or intracytoplasmic sperm injection in our hospital. INTERVENTION(S): Embryos, at the cleavage stage, were cryopreserved using the vitrification method in either a plastic straw or a nylon mesh container. The embryos were thawed using an initial osmotic pressure of either 0.5 M or 1.0 M sucrose with subsequent step-wise dilution. After thawing, the embryos were transferred to the uterus. MAIN OUTCOME MEASURE(S): Survival rate of blastomeres, embryo survival rate, implantation, and pregnancy rates, cancellation rate because of embryo damage. RESULT(S): Use of nylon mesh and the 1.0 M sucrose thawing solution significantly improved blastomere survival rate (98.0 +/- 1.0%, mean +/- SEM), pregnancy rate (41.0%) and implantation rate (32.3%). CONCLUSION(S): Vitrification using a nylon mesh container and subsequent thawing in a 1.0 M sucrose solution is an easy and inexpensive method that improves the reliability of embryo cryopreservation of embryos without adverse effects on clinical outcomes.

[Aging and anti-aging of oocytes].

The functions of organs decrease as the age increases. The fecundity of women also decreases due to mainly the decreasing quality of oocytes. The recent change of life style makes the age of infertility patients elder and infertility treatments more difficult. The therapeutic strategy for the elder infertility patients is still chaotic. The precise evaluation for the ageing in the female genital organs should be developed and the treatment for the failure of fecundity due to aging must be overcome.

Distribution of CESP-1 protein in the corneal endothelium and other tissues.

PURPOSE: The gene expression profile of human corneal endothelium (CE) was established with the gene signature system. A novel gene, GS3582, was abundantly transcribed in the CE compared with other tissues according to a human gene expression database. This protein was designated corneal endothelium-specific protein (CESP)-1. The tissue distribution and subcellular localization of CESP-1 was assessed in humans and mice, to investigate its physiological function. METHODS: Rabbit and mouse CESP-1 cDNAs were cloned, and a polyclonal anti-human CESP-1 antibody (Ab) and anti-mouse N- or C-terminal ovary-specific acidic protein (OSAP)-1 Ab were produced. CESP-1 expression was investigated in human and mouse corneas by Western blot and/or immunohistochemical analysis. The distribution of CESP-1 in human tissues was also examined by Western blot analysis. To identify the subcellular localization of CESP-1, cultured human CE was colabeled with anti-human CESP-1 Ab and anti-cytochrome c monoclonal Ab or anti-GRP78 monoclonal Ab for confocal microscopy. RESULTS: The rabbit and mouse CESP-1 cDNA sequences contained an open reading frame coding 242 and 283 amino acids, respectively. Mouse CESP-1 was entirely consistent with mouse OSAP. Western blot analysis showed that CESP-1 was expressed in the human corneal epithelium, CE, cultured CE, brain, testis, and ovary. Mouse CESP-1 was also expressed in mouse corneal epithelium and CE with anti-mouse C- but not N-terminal OSAP Ab according to immunohistochemical analysis. Subcellular localization of CESP-1 to the mitochondria was demonstrated in cultured human CE. The N-terminal of CESP-1, possessing a mitochondrial targeting sequence, may be processed after the protein is imported into the mitochondria. CONCLUSIONS: CESP-1 was distributed in the corneal epithelium, the CE and cultured human CE, as well as the brain, testis, and ovary. CESP-1 was localized in the mitochondria of cultured human CE. These findings may provide some clues about the physiological function of CESP-1.